The release of neurotransmitters at synapses is a very important process that is responsible for information flow through brain circuits. Modifications in neurotransmitter release also are likely to be involved in changing neuronal function during development, learning, disease states, and other forms of brain plasticity. Neurotransmitter release is known to result from the orderly conduit of synaptic vesicles through a series of membrane trafficking reactions. The general goal of this project is to understand the molecular basis for these reactions, in particular the exocytotic release of neurotransmitters and the endocytotic recycling of vesicle components. In recent years, many presynaptic proteins have been identified and a large fraction of these now have been implicated in neurotransmitter release. However, because so many proteins are involved, it is difficult to sort out the specific role that each plays in synaptic vesicle trafficking. We will address this problem by defining the temporal order of protein action, specifically by determining when several key proteins act relative to each other and relative to the time at which synaptic vesicles fuse or are endocytosed. For this purpose, we will use high-resolution optical methods to determine the timing of protein action. The proteins to be considered include SNARE proteins, clathrin, and clathrin-associated proteins, such as AP180, Eps15, epsin, and auxilin. Flash photolysis of caged binding-site peptides will be used to examine several interactions involving SNARE proteins, while Fluorescence resonance energy transfer will be used to look at interactions of clathrin with its banding partners during endocytosis. The results of these experiments will discriminate among many existing molecular models of synaptic vesicle trafficking reactions and lead to more refined, quantitative models. By clarifying several important aspects of the molecular basis of synaptic communication in the brain, this work will ultimately yield insights into the etiology of numerous neurological disorders that result from defects in synaptic transmission.